The goal of this project is to 1) understand the role of mitochondria in ischemia-reperfusion injury and cardioprotection; 2) to understand the role of altered ion homeostasis and altered metabolism in ischemia-reperfusion and cardioprotection and 3) to understand changes in cytosolic and mitochondrial signaling involved in cardioprotection and cell death. It is proposed that ischemic preconditioning (PC) initiates signaling that converges on mitochondria and results in cardioprotection. We also examined mechanisms by which inhibition of glycogen synthase kinase (GSK) mediates cardioprotection by acting on the mitochondria. Inhibition of GSK-3 reduces ischemia-reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to determine the molecular targets and mechanistic basis of this cardioprotective effect. We found that GSK inhibition slowed ATP consumption under these conditions, which could be due to inhibition of ATP entry into the mitochondria through VDAC and/or ANT or to inhibition of the F1F0 ATPase. We therefore used proteomic methods to test the hypothesis that PC and pharmacological preconditioning similarly alter mitochondrial signaling complexes. Langendorff-perfused murine hearts were treated with a specific GSK-3 inhibitor AR-A014418 (GSK Inhib VIII) for 10 min or subjected to 4 cycles of 5-min ischemia/reperfusion (PC) prior to 20-min global ischemia and 120-min reperfusion. PC and GSK Inhib VIII both improved post-ischemic left ventricular developed pressure (LVDP) recovery, decreased infarct size, and reduced lactate production during ischemia compared to their time-matched controls. We used proteomics to examine mitochondrial protein levels/post-translational modifications that were common between PC and GSK Inhib VIII. Levels of cytochrome c oxidase subunits Va and VIb, ATP synthase-coupling factor 6, and cytochrome b-c1 complex subunit 6 were increased while cytochrome c was decreased in PC and GSK Inhib VIII. Furthermore, the amount of cytochrome c oxidase subunit VIb was found to be increased in PC and GSK Inhib VIII mitochondrial supercomplexes, which are comprised of complexes I, III, and IV. This result would suggest that changes in complex subunits associated with cardioprotection may affect supercomplex composition. Thus, the ability of PC and GSK inhibition to alter the expression levels of electron transport complexes will have important implications for mitochondrial function. Isolated mitochondria from mice deficient in cyclophilin D (CypD-/-) are less sensitive to Ca2+-induced opening of the mitochondrial permeability transition (MPT) in vitro. Thus, the lack of CypD enables heart mitochondria to take up more Ca2+ before undergoing the MPT. We hypothesize that the MPT serves as a Ca2+-safety valve that can open to release excess Ca2+, but not necessarily result in death. If the MPT is blocked in CypD-/- mice, we hypothesize that matrix Ca2+ (Ca2+m) would be higher in CypD-/- mice compared to WT and this would activate Ca2+-sensitive NADH dehydrogenases (e.g., pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH)), which would in turn, alter oxidative metabolism and increase oxygen consumption. Consistent with this, we found altered expression levels of PDH E1 subunit and the alpha-KGDH E2 subunit in CypD-/- hearts using 2D DIGE proteomics. To evaluate differences in metabolism, we perfused hearts with 13C-glucose and 13C-palmitate and looked at their contribution to the acetyl-CoA pool by measuring label incorporation into the C4 of glutamate. The 13C-labeled glucose or palmitate enters the Krebs cycle and labels the alpha-KG pool that is in equilibrium with glutamate, which is usually present at higher levels. The ratio of glucose to palmitate metabolism in CypD-/- hearts was 1.5-fold higher than in WT, which would suggest increased PDH activity. 13C-labeling into succinate compared to glutamate was also increased significantly in CypD-/- hearts, and this result would be consistent with increased activity of alpha-KGDH relative to other competing reactions. We measured alpha-KGDH activity to evaluate whether Krebs cycle flux upstream of succinate was elevated in CypD-/- hearts and found a 1.4 fold increase in alpha-KGDH activity. Therefore, these results demonstrate that the loss of a MPT component, CypD, results in physiological flux changes in the Krebs cycle and oxidative metabolism that are consistent with increased Ca2+m.